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<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Hi Marina<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Just a follow up to my previous reply. I
notice you are running the sample in 5% glycerol? The viscosity of this alone
is 1.127E-2, not what you have mentioned in your mail. If this is the case then
this would alter the S20,w of your protein substantially. <o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Thanks<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Tom<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
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face="Times New Roman"><span style='font-size:12.0pt'>
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<p class=MsoNormal><b><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma;font-weight:bold'>From:</span></font></b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>Thomas Jowitt<br>
<b><span style='font-weight:bold'>Sent:</span></b> 03 March 2008 10:17<br>
<b><span style='font-weight:bold'>To:</span></b> Fasolini, Marina [Nervianoms];
rasmb@rasmb.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> RE: [RASMB] problem with
fitting</span></font><o:p></o:p></p>
</div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Dear Marina<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>First of all, unless your gel filtration
is linked to a light scattering module to estimate the absolute molecular mass,
then I wouldn’t trust the estimated molecular weight from a calibrated column.
A sedimentation value of 6.23 is most likely to be around 150kDa unless the
molecule is substantially elongated, as a hexameric molecule of this size and
sedimentation value would have a frictional ratio of around 2.5 (certainly not
impossible). <o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>The two experiments that I would perform
to answer these questions are, firstly an equilibrium experiment to establish
the correct molecular weight, done with at-least three concentrations to
establish if oligomerisation is concentration dependant. Secondly, I would run
the crystal structure through a modeling program such as SOMO to generate a
bead model, or hydropro to create a shell around the crystal structure. Either
of these would give you a decent estimate of the sedimentation value expected
for a rigid molecule of that size. <o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Thanks<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Tom Jowitt<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><b><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma;font-weight:bold'>From:</span></font></b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>Fasolini, Marina [Nervianoms]<br>
<b><span style='font-weight:bold'>Sent:</span></b> 03 March 2008 09:10<br>
<b><span style='font-weight:bold'>To:</span></b> rasmb@rasmb.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> [RASMB] problem with
fitting</span></font><o:p></o:p></p>
</div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Dear AUC users,</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I have a problem in the fitting of some data and I would
like to have your opinion.</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I want to control the sedimentation of a protein
in order to define the state of oligomerization. It is published as
forming an hexameric homo oligomer. From the crystal
structure, it is a wheel like structure of 160 angstrom in
diameter, with a central role of 15 angstrom in diameter. The thickness is
20-40 angstrom. How can I expect it to sediment? How can I improve my parameters
or model in order to improve the fitting?</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>The protein is 50KDa but in gel filtration
it cames out as hexamer (300 KDa). In my sedimentation
experiment I see one peak of 6.23S which corresponds to
140KDa. <font color=blue><span style='color:blue'>Fitting of the
sedimentation data was done with SedFit. </span></font>Do you think I can
say that it is a trimer? I would expect it as an hexamer.</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I hope someo<font color=blue><span style='color:blue'>ne can</span></font>
help me<font color=blue><span style='color:blue'>. </span></font></span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>the conditions are : </span></font><o:p></o:p></p>
</div>
<div>
<div u1:shape="_x0000_s2050">
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Vbar 0.7397 </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Viscosity 1.02530 </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Density 0.01183 </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Thanks a lot</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><st1:place w:st="on"><st1:City w:st="on"><font size=2
face=Arial><span style='font-size:10.0pt;font-family:Arial'>Marina</span></font></st1:City></st1:place><font
size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
</div>
<div>
<p class=MsoNormal><font size=2 face="Times New Roman"><span style='font-size:
10.0pt'> </span></font><o:p></o:p></p>
</div>
</div>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>MARINA FASOLINI</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Structural Chemistry </span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'><a href="http://www.nervianoms.com/">Nerviano Medical
Sciences</a></span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Viale Pasteur 10</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>20014 Nerviano - Milano</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'><a href="mailto:marina.fasolini@nervianoms.com">marina.fasolini@nervianoms.com</a></span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Tel. +390331581462</span></font><o:p></o:p></p>
</div>
<div>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Fax. +390331581360</span></font><o:p></o:p></p>
</div>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
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